Collagens are the most abundant proteins in the animal kingdom. Twenty-five different types are currently known. The basic structural unit is a triple helix; in collagen I, the helix consists of three polypeptides, each containing 1050 amino acids. Collagen fibrils form by lateral interactions between the triple helices. Some collagens, notably collagen IV, form two-dimensional sheets.
The amino acid sequences of collagen molecules are highly repetitive, and this regularity is reflected in the structure of collagen fibrils. The amino acid sequence of collagen I contains about 20 copies of an 18-amino acid motif in which every third amino acid is a glycine.
The various collagens are produced by fibroblasts and some epithelial cells. The original transcript is a pro-collagen polypeptide that contains signal sequences for export from the cell and also a pro-peptide that prevents association to form triple helices. About 50% of proline residues and 15-20% of the lysines in pro-collagen chains are subject to intracellular processing to form hydroxyproline and hydroxylysine. These modifications are essential for the mechanical properties of collagen. Outside the cell, the pro-peptides are cleaved off, starting the process of self-assembly.
Collagens are essential components of structures such as bones and tendons and also of extra-cellular matrix in general. For instance, collagen IV forms the basic network of the basement membranes to which epithelial and endothelial cells attach. Part of the diversity of collagens is explained by the different types of collagen, but there is also a large variety of collagen-associated molecules. Collagen fibres are usually associated with proteoglycans. These proteins, consisting of a core polypeptide and one or more glucosaminoglycan side chains, are also a very diverse class. In the basement membrane, laminin and entactin (nidogen) are important components. The fibulins are a class of proteins with binding sites for several basement membrane proteins. Undulin is a fibre-forming protein that is found in association with collagen in low amounts in normal liver, and in high amounts in fibrotic liver.
Collagen and other proteins in connective tissue contain the Arg-Gly-Asp amino acid sequence, which confers binding to the integrin class of cell adhesion molecules. Other amino acid sequences may also constitute the core binding motif, and other parts of the ligand contribute to affinity as well as specificity. The β1 integrins are important in collagen binding. Other collagen-binding proteins include the discoidin domain receptors, which respond to collagen by activating a tyrosine kinase.
Collagen fibres are laterally flexible, but neither elastic nor compressible. Elastic properties of connective tissue are contributed by the protein elastin and its associated proteins oxytalan and elaunin. Fibrillins 1 and 2 are other proteins that form elastic fibres in association with elastin and another structural component, microfibril-associated glycoprotein. Abnormal elastic fibres are found in areas of hepatic fibrosis.
Deposition of collagen is a common process in healing of injury, leading to the formation of the familiar “scar tissue”. Collagen deposition is a process that decreases the functionality of the tissue. This is obvious where elasticity of the tissue is important, a salient example being the scar tissue that forms during healing of a myocardial infarction. In the liver, the effects of rigid fibres are less obvious. Part of the process of liver fibrosis is deposition of extra-cellular matrix material in the space between the hepatocytes and the fenestrated endothelium of hepatic sinusoids, coincident with the transformation of sinusoids into capillaries that have an ordinary basement membrane. This transformation diminishes the functionality of the liver by impeding the transfer of solutes between the blood and the hepatocytes.
Hepatic fibrosis starts with injury that causes damage or death of liver cells. The injury initiates an inflammatory response. Release of cytokines, chemotactic factors and fragments of ECM matrix proteins (collagen and fibronectin) cause activation of liver cells and recruitment of inflammatory cells, such as granulocytes. Inflammation, including oxidative stress, is the common factor of most causes of hepatic fibrosis. An important event is the activation of stellate cells (aka fat-storing cells or Ito cells). The best-known function of these cells in the normal liver is to store vitamin A. On activation, they lose their vitamin A and differentiate into myofibroblasts. These cells are the collagen-producing cells.
Causative agents of liver fibrosis are numerous: alcohol, hepatitis viruses, cholangitis, hemachromatosis, Wilson's disease and schizostomiasis. In experimental animals (usually rats), fibrosis may be induced by carbon tetrachloride or thioacetamide. Most of these agents produce distinct patterns of liver injury, including collagen deposition. The role of the inflammatory response is variable; in some conditions, for instance hemochromatosis, oxidative stress is important.
If the injury is limited in extent and in time, the resulting fibrosis is reversible. In the liver, prolonged stress may lead to cirrhosis, characterised by general damage, formation of regeneration nodules, and fibrosis that distorts liver architecture. In rats, collagen Type I have a half-life of 30 days and Type III has a half-life of 15 days. When cirrhosis is induced by carbon tetrachloride, the half-lives of both collagens are reduced by 50%. Amounts of collagen reach levels 5-10 times higher than normal values (but never above 30-35 mg/g).
The point of diagnosis and treatment of hepatic fibrosis is the prevention of irreversible liver damage and consequent reduced function. Increased amount and altered patterns of collagen deposition indicate liver fibrosis. A positive biopsy is considered the definitive answer. Biopsies are invasive procedures with a frequency of significant complications of 1-5%. Single unguided biopsies will miss cirrhosis in 10-30% of cases. Correct diagnosis may increase to 100% if three specimens are examined. As the incidence of complications increases with the number of biopsies taken, it appears that triple biopsies may increase the incidence of complications to the order of 10%. Furthermore, evaluation of biopsies is far from straight-forward.
Within any stage of liver disease, there is up to a four-fold variation in the area of fibrosis; furthermore, there is a substantial overlap in the area of fibrosis between different stages. Consequently, the amount of collagen, as calculated by computer-aided image analysis, is of little value in deciding the stage of fibrosis. Experienced observers using standardised scoring schemes do provide reliable information on staging. In fact, these systems work so well that there is little incentive to look beyond collagen for additional histological markers. However, the ECM matrix protein tenascin is deposited in early lesions and is often absent from mature scar tissue, while vitronectin is a marker of mature fibrous tissue.
Serological markers for liver fibrosis that are used up to now may be divided in two groups: Markers for alterations in hepatic function (platelet counts, liver trans-aminases) and markers for ECM turnover. The latter may include markers of collagen deposition (e.g., circulating collagen pro-peptides) and/or collagen degradation (e.g., circulating fragments of collagen IV). A combination of carefully selected markers may give much more precise results than single markers, but there is no universal agreement on this issue.
There is clearly a need for a reliable test that can diagnose fibrosis in the early stages, before irreversible damage occurs. A very desirable feature of future tests is the ability to quantify changes in the ECM.
As explained above, excessive deposition of collagen reduces the elasticity of the tissue. This also includes the scar tissue that forms during healing of a myocardial infarction. Following injury to the heart or persistent increase in stress in the cardiac wall, the heart attempts to compensate by remodelling. This process implies progressive alterations in the size and shape of the ventricular chambers, coupled with changes in the composition of the myocardium. Typical responses include enlargement of surviving myocytes and changes in the types, cross-linking and concentration of collagen. It appears that initially, the extra-cellular matrix is partially degraded concurrent with hypertrophy of cardiac myocytes. Subsequently, there is a chronic compensatory phase as the collagen concentration returns to normal. But if the heart is unable to compensate, remodelling results in marked ventricular dilatation in spite of prominent fibrosis. The end stage of cardiac failure is characterised by further remodelling of the extra-cellular matrix concurrent with disorganisation of myofibrils and loss of myocytes. Collagen continues to accumulate, but collagen fibrils are laid down in an irregular manner.
Fibrosis is a component of more than 200 lung diseases. Repeated injury or sustained stress e.g., inflammation and/or inhaled particles, are common components of the etiology, along with genetic factors that turn the balance in the direction of deposition of connective tissue. One example is deficiency of α1-proteinase inhibitor, a protein whose activity may also be reduced as a consequence of smoking. Its main function is to inhibit neutrophil elastase. As in fibrosis of other organs, imbalance between synthesis and degradation may initiate repair processes that actually injure function. As in fibrosis of the heart or the liver, myofibroblasts figure prominently in the pathology. They are originally recruited as fibroblasts that subsequently differentiate. It appears that the “repair process” may continue in the absence of perceptible inflammation, resulting in progressive loss of function.